8 replies to this topic

[Travis]

Hi, I am currently doing luciferase assay. I need to transfect B-gal, PGL-3 construct, PCDNA3 and some other vectors to MDA-MB-231 and T47D human breast tumor cell lines. I have been doing it with the MCF-7 cell line last week and it was totally fine. However this week I follow the same steps with the MBA-MD-231 and T47D cell lines but then I cant get the data. There just simply doesnt have any luciferase and B-gal protein in the cells. What are the possible problems in this? Is it because of low transfection efficiency? How can I optimize the experiment so I can get some data? Thanks very much!!

[genehunter]

I assume you used the same ratio of total DNA to whatever the transfection agent that you are using for both experiments. I dont have any experience with T47D, but MBA-MD-231 is as readily transfectable as MCF-7 on my hands. Be sure the toxicity is not overwhelming and the cell density is not too high. You can optimize it with fixed amount of cmv-luc vector or egfp vector, change the ratio of vector to transfection reagent. Use cells that are 70%-90% confluent at the time of transfection.

[Curtis]

I also used lucierase vectors with pcDNA3 and transfected into MCF7....

the process was very easy and I did calcium-phosphate transfection (not lipofectamin) ....in the end I did freeze-thaw to break the cells and extract the proteins (not any lysis buffer)...then I read the signal by a luminometer

I have the protocol somewhere, if you need it just let me know!

[Travis]

Curtis, on Mar 7 2009, 10:08 PM, said:

I also used lucierase vectors with pcDNA3 and transfected into MCF7....

the process was very easy and I did calcium-phosphate transfection (not lipofectamin) ....in the end I did freeze-thaw to break the cells and extract the proteins (not any lysis buffer)...then I read the signal by a luminometer

I have the protocol somewhere, if you need it just let me know!

Oh thank you! can you please give me the protocol?? I would love to give it a try... so I just need calcium phosphate to do the transfection?

[NemomeN]

Not all cells are transfectable with the same efficiency (or even at all). If you still have problems, you can try nucleofection (Amaxa)

[Curtis]

pm your e-mail to me because I can't send message to you, it says you have disabled your message service or something

Edited by Curtis, 09 March 2009 - 01:46 AM.

[cotchy]

Curtis could i get a copy of that protocol from you to?

Thanks very much.

Cotchy.

[Travis]

genehunter, on Mar 7 2009, 02:16 PM, said:

I assume you used the same ratio of total DNA to whatever the transfection agent that you are using for both experiments. I dont have any experience with T47D, but MBA-MD-231 is as readily transfectable as MCF-7 on my hands. Be sure the toxicity is not overwhelming and the cell density is not too high. You can optimize it with fixed amount of cmv-luc vector or egfp vector, change the ratio of vector to transfection reagent. Use cells that are 70%-90% confluent at the time of transfection.

Hi, may I ask what tranfection agent you use for your experiment?? I am currently using Polyfect from Qiagen and it doesnt work for MDA-MB231, but its alright for MCF-7. Thanks

[Curtis]

cotchy, on Mar 9 2009, 02:41 AM, said:

Curtis could i get a copy of that protocol from you to?

Thanks very much.

Cotchy.

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